U.S. patent application number 16/604465 was filed with the patent office on 2020-05-14 for cosmetic preparation containing white truffle extract and cosmetic method thereof.
This patent application is currently assigned to ISP Investments LLC. The applicant listed for this patent is ISP Investments LLC. Invention is credited to Rachel CHABERT, Corinne COQUET.
Application Number | 20200146971 16/604465 |
Document ID | / |
Family ID | 62002122 |
Filed Date | 2020-05-14 |
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United States Patent
Application |
20200146971 |
Kind Code |
A1 |
COQUET; Corinne ; et
al. |
May 14, 2020 |
COSMETIC PREPARATION CONTAINING WHITE TRUFFLE EXTRACT AND COSMETIC
METHOD THEREOF
Abstract
The present invention provides a process for obtaining an
extract from White Truffle (Tuber magnatum) for skin cosmetic use,
the white truffle extract obtainable by the process and a cosmetic
composition comprising the said extract. The invention provides a
method for reducing the signs of aging and lightening the skin.
Inventors: |
COQUET; Corinne; (CIPIERES,
FR) ; CHABERT; Rachel; (Grasse, FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ISP Investments LLC |
Wilmington |
DE |
US |
|
|
Assignee: |
ISP Investments LLC
Wilmington
DE
|
Family ID: |
62002122 |
Appl. No.: |
16/604465 |
Filed: |
April 10, 2018 |
PCT Filed: |
April 10, 2018 |
PCT NO: |
PCT/EP2018/059190 |
371 Date: |
October 10, 2019 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61Q 19/02 20130101;
A61K 8/9728 20170801; B01D 11/0288 20130101; B01D 11/0257 20130101;
B01D 11/02 20130101; A61Q 17/04 20130101; A61Q 19/08 20130101; A61Q
19/00 20130101 |
International
Class: |
A61K 8/9728 20060101
A61K008/9728; A61Q 19/08 20060101 A61Q019/08; A61Q 17/04 20060101
A61Q017/04 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 14, 2017 |
CN |
201710244119.7 |
Claims
1. A process for obtaining an extract from white truffle (Tuber
magnatum) comprising: (i) adding water to grounded white truffles
to make a mixture, (ii) agitating the said mixture by maintaining
the temperature from RT to 50.degree. C. (iii) filtering the
mixture to remove the solid part to obtain the extract.
2. The process according to claim 1, wherein step (ii) is made at a
temperature of 50.degree. C.
3. The process according to claim 1, wherein said extract of step
(iii) is further clarified by sequential filtration from 50 .mu.m
to 20 .mu.m decreasing porosity until 0.5 .mu.m to 0.2 .mu.m to get
the extract.
4. The process according to claim 1, wherein said white truffle
extract of step (iii) is furthermore diluted at a concentration
between 0.5 g/Kg and 1.5 g/Kg, of dry matter in a solvent selected
from water, glycerol, ethanol, propanediol, butylene glycol,
dipropylene glycol, ethoxylated or propoxylated glycols, cyclic
polyols or any mixture of these solvents.
5. A white truffle extract obtained by the process of claim 1.
6. The white truffle extract of claim 5, wherein the said white
truffle extract comprises from 0.5 to 1.5 g/kg of dry matter, from
0.05 to 0.15 g/kg of protein, from 0.35 to 1.05 g/kg of sugar, from
0.025 to 0.075 g/kg of amino acids; and from 0.05 to 0.15 g/kg of
phenolic compounds.
7. The white truffle extract of claim 5, comprising compounds
having a molecular weight of less than 60 kDa.
8. A cosmetic composition comprising a white truffle extract
obtained according to the process of one of the claims 1 to 4,
wherein the said white truffle extract comprises compounds having a
molecular weight of less than 60 kDa and a physiologically
acceptable medium.
9. The cosmetic composition of claim 8, wherein said white truffle
extract is used in a concentration from about 0.01% by weight to
about 20% by weight of the total weight of the composition.
10. The cosmetic composition of claim 8, wherein said white truffle
extract is used in a concentration from about 0.1% by weight to
about 5% by weight of the total weight of the composition.
11. The cosmetic composition of claim 8, wherein said composition
is in a form adapted for topical application.
12. The cosmetic composition of claim 8, wherein said composition
is in the form of gels, creams, balms, milks or foaming
products.
13. The cosmetic composition of claim 8, further comprising at
least one other active agent.
14. The cosmetic composition of claim 8, further comprising at
least one other active agent selected from antioxidant agent,
hydrolysate of plants, synthetic peptide compound, sunscreen and
anti-wrinkle agent.
15. A method for reducing and/or correcting the signs of aging and
photo-aging of the skin, comprising topically applying to the skin,
a cosmetic composition comprising the white truffle extract
obtained according to the process of one of claim 1.
16. A method for lightening the skin, comprising topically applying
to the skin, a cosmetic composition comprising the white truffle
extract obtained according to the process of one of claim 1.
Description
TECHNICAL FIELD
[0001] The present invention is in the field of cosmetics and more
specifically in the field of skin care. The invention relates to a
process for preparing a white truffle (Tuber magnatum) extract by
aqueous extraction, a white truffle extract obtainable by the
process and a cosmetic composition comprising the white truffle
extract.
[0002] The invention also relates to a method for reducing and/or
correcting the signs of aging and photo aging of the skin, and to
lighten the skin.
[0003] The white truffle extract can be used alone or in
combination with other active agents.
BACKGROUND
[0004] The skin is a vital organ composed of several layers
(dermis, proliferative layers and stratum corneum), which covers
the entire surface of the body and ensures protective, sensitive,
immune, metabolic or thermoregulatory functions. The skin, like the
other organs, is subject to aging.
[0005] For example, the appearance of the skin is modified by
various types of internal (disease and hormonal changes such as
pregnancy) or external aggressions (environmental factors, such as
pollution, sunlight, ultraviolet radiation, pathogens, etc.). Then
wrinkles and fine lines, hyperpigmentation or hypopigmentation
blemishes, dryness or even dehydration of the skin, thinning of the
epidermis, elastosis, imperfections, age spots, etc., may
appear.
[0006] In the skin, premature aging is observed, occurring in the
areas exposed to ultraviolet radiation.
[0007] With age, skin appearance is altered. Dark spots, dull
complexion, loss of tone uniformity are noticeable effect
associated with age. The loss of skin youthful glow is often one of
the first extrinsic signs of aging and constant exposure to the UV
rays of the sun also causes the skin to appear dull and dry.
[0008] It is known that fungi are a wide family of living
organisms. Fruiting bodies of some wild and cultivatable mushrooms
contain medicinal compounds which are being used in traditional
medicines and cosmetics. Fungi have developed during their
evolution various properties to resist environmental stress and it
is known that some fungi compounds can have an effect to moderate
damages caused by life circumstance and natural environment on
organisms. Thus, research for new natural bioactive compounds from
fungi origin has intensified. For example, Inonotus obliquus
mushroom contains enzyme such as Superoxide Dismutase (SOD), which
is a key element in protecting the cell against ROS.
[0009] Also, truffles are known to be effective on certain
conditions such as the improvement of immunity and hyperlipidemia.
The genus Tuber belongs to Ascomycete phylum and is subterranean
fungus. The Tuber fungi genus comprises some species of truffles.
These truffle species are hypogenous fungi that establish an
ectomycorrhizal symbiosis with trees and shrubs and form a fruiting
body.
[0010] Some of the truffle species are highly priced as a food, due
to their fruiting bodies characteristic aroma and delicious taste.
These species are referred to by "truffles" or "real truffles" and
they are precious and expensive delicacies which are widely used in
the famous French and Italian cuisines.
[0011] Common truffles, which belong to the class of ascomycetes,
order of Tuberales, have been appreciated as excellent edible fungi
for many centuries and are of outstanding importance due to their
special aroma. Truffles are underground fungi growing in symbiosis
with oak tree roots and forming tuber-like fruiting bodies. In the
extraction process of truffles, several active agents are set free.
It has been found that a non-specific stimulation of the immune
system can be achieved.
[0012] In the description of the invention "Truffles" refers to the
following species: The Italian white truffles (scientific name:
Tuber magnatum pico), the black truffle (also known as Perigord
truffles from France, scientific name: Tuber melanosporum), the
Burgundy truffle (Tuber uncinatum), Kalahari truffle (Terfezia
pfeilii), Lion-Truffle (Terfezia leonis), summer truffle (Tuber
aestivum), winter truffle (Tuber brumale), Chinese Truffle (Tuber
sinensis or Tuber indicum) and Bianchetto or whitish truffle (Tuber
borchii).
[0013] The white truffle (Tuber magnatum) is referred as Piedmont
or Alba Truffle. Truffles grow a few inches down in the earth, in
symbiosis with the roots of hardwood trees like oaks, chestnut,
hazelnut and hornbeam. The irregularly shaped knobby little spheres
range in size from around an inch and may weight over a pound
(though truffles of that weight are rare). The firm flesh of a
white truffle is pale cream to light brown in color, with white
marbling throughout. Truffles are the true fruit of the earth,
rarer and precious than any other edible root, tuber or mushroom.
There is no other flavor like them on earth, which is perhaps why
they are so often described as heavenly.
[0014] There have been many attempts to domestic these wild
truffles, though they can take a decade to grow. Some of these
attempts have been successful, but the most reliable source is
still to forage for them in nature.
[0015] From the state of the art, numerous cosmetics are known
which in some way contain plant-based raw materials in the form of
oils or extracts. In most cases, the known advantageous effects of
individual plants are used to achieve a corresponding overall
effect.
[0016] The use of white truffle extract in a cosmetic composition
is known in prior art. Most extract are total extracts or
water-alcohol extracts. For example, the Chinese patent
application, CN-A-104856928 disclosed an anti-wrinkle cosmetic
wherein the active substance is one of mixture A, Echinaceapurpurea
extract, and white truffle extract, or any combination thereof, and
said mixture A is a mixture of trifluoroacetyl tripeptide-2,
glycerol and dextran. U.S. Pat. No. 6,843,995 discloses a cosmetic
preparation comprising of at least one aqueous extract of common
truffles (Tuberacea) together with spray-dried champagne, wherein
the active complex is provided in a cosmetic acceptable gel and
stabilizer. Chinese patent CN-A-104666237 discloses a preparation
method using hydro-alcoholic extract and the application of the
truffle active ingredient for preparing an antioxidant and a skin
whitening product. However, no document disclosed a water
extracting method to prepare an entirely safe and effective
cosmetic ingredient from white truffle.
[0017] Despite the various anti-aging cosmetic products on the
market for the treatment of skin, there remains a need for
effective topically applied cosmetic compositions that provide
anti-aging or rejuvenating benefits to the skin, hair and/or nails
using natural ingredients as active agent. Unnatural,
chemically-synthesized products may be perceived as being
environmentally or personally unsafe. In contrast, natural products
are perceived as pure, mild, and superior to chemically synthesized
products. Numerous natural based products extracted from plants or
herbs are known to contain antioxidant/free-radical scavenging
agents that can neutralize the effects of free-radical damage.
Additionally, they can contain agents that stimulate the synthesis
and restoration of damaged connective tissue structures in the
dermis and barrier function in the epidermis.
[0018] There remains a need for cosmetic compositions which address
the signs of aging, in particular the appearance of wrinkles,
lines, and sagging. It is therefore an object of the present
invention to provide new compositions and methods for treating,
ameliorating, and/or preventing signs of aged or aging skin. It is
a further object of the invention to improve the overall appearance
of aging or aged skin.
[0019] The foregoing introduction is presented solely to provide a
better understanding of the nature of the problems confronting the
art and should not be construed in any way as an admission as to
prior art nor should the citation of any reference herein be
construed as an admission that such reference constitutes "prior
art" to the instant application.
SUMMARY
[0020] The main aspect of the present invention provides a process
for obtaining an extract containing fruiting body of a mushroom
(White Truffles) of the genus Tuber magnatum, comprising the
following steps: [0021] (i) adding water to white truffles to make
a mixture, [0022] (ii) agitating the said mixture by maintaining
temperature from RT (Room Temperature) to below 80.degree. C.
[0023] (iii) filtering the mixture to remove the solid f part to
obtain the extract.
[0024] In another aspect, the present invention provides a white
truffle extract obtained by the process of the present
invention.
[0025] In another aspect, the present invention provides a cosmetic
composition comprising white truffle extract obtained by aqueous
extraction, wherein the white truffle extract comprises compounds
having a molecular weight of less than 60 kDa in a physiologically
acceptable medium.
[0026] In yet another aspect, the present invention provides a
method for reducing and/or correcting the signs of aging and
photo-aging of the skin, comprising topically applying to the skin
a cosmetic composition comprising white truffle extract obtained by
aqueous extraction, wherein the white truffle extract comprises
compounds having a molecular weight of less than 60 kDa in a
physiologically acceptable medium.
[0027] In yet another aspect, the present invention provides a
method for reducing skin pigmentation and/or lightening the skin,
comprising topically applying to the skin a cosmetic composition
comprising white truffle extract obtained by aqueous extraction,
wherein the white truffle extract comprises compounds having a
molecular weight of less than 60 kDa in a physiologically
acceptable medium.
BRIEF DESCRIPTION OF THE DRAWINGS
[0028] Further embodiments of the present invention can be
understood with the appended figures.
[0029] FIG. 1 is illustration of collagen I expression on
fibroblasts assessed by immunofluorescence.
[0030] FIG. 2 is illustration of melanin content on ex vivo skin
biopsies (Fontana-Masson).
[0031] FIG. 3 is illustration of stimulation of LC3 (autophagy
pathway), evaluated on keratinocytes.
[0032] FIG. 4 is illustration of stimulation of LC3 (autophagy
pathway), evaluated on ex vivo skin biopsies.
DETAILED DESCRIPTION
[0033] Detailed embodiments of the present invention are disclosed
herein; however, it is to be understood that the disclosed
embodiments are merely illustrative of the invention that may be
embodied in various forms. Therefore, specific structural and
functional details disclosed herein are not to be interpreted as
limiting, but merely as a representative basis for teaching one
skilled in the art to variously employ the present invention.
[0034] Whenever a term is identified by reference to a range, the
range will be understood to explicitly disclose every element
thereof. As a non-limiting example, a range of 1-10% will be
understood to include 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, and 10%,
and all values between 1 and 10%.
[0035] Where two or more substituents are referred to as being
"selected from" a group of enumerated alternatives, it is meant
that each substituent can be any element of that group, independent
of the identity of the other substituents.
[0036] As used herein, "% refers % by weight, that is the weight
percent of a component in relation to the total weight of the
composition (i.e., including any carriers, vehicles, solvents,
fillers, or other components added before application to the skin)
unless otherwise provided.
[0037] All terms used herein are intended to have their ordinary
meaning unless otherwise provided. For the purposes of describing
and claiming the present invention, the following terms are
defined:
[0038] "Extract" is understood to be any substance or isolated
preparation extracted from a natural source, regardless of
extraction method or ingredients. The term is used in a broad sense
including, for example, ingredients soluble in water or an organic
solvent extracted from a natural substance using the solvent, or
specific ingredients of a natural substance.
[0039] "Aqueous extract" is understood to be a mixture of compounds
obtained by extraction with water.
[0040] It is understood by "physiologically acceptable" that the
white truffle extract according to the invention, or a composition
containing said agent, is suitable for coming into contact with the
skin or a mucus membrane without provoking a toxicity or
intolerance reaction.
[0041] "Cutaneous signs of aging and photo-aging" refers to all
changes in the external appearance of the skin and keratinous
appendages due to aging, such as, for example, thinning of the
skin, sagging, loss of hydration and atonia, deep wrinkles and fine
lines, loss of firmness, dullness, dermal atrophy or any other
internal degradation of the skin resulting from exposure to
ultraviolet radiation such as age spots.
[0042] "skin lightening" refers to the improvement in skin tone,
radiance, and/or clarity and/or restoration of skin luster or
brightness.
[0043] The species name "Tuber magnatum", or "white truffle" or
"trifola d'Alba Madonna" ("Truffle of the White Mother" in Italian)
is found mainly in the Langhe and Montferrat areas of the Piedmont
region in northern Italy and, most famously, in the countryside
around the cities of Alba and Asti. White truffle is characterized
by a specific smell very fine and rare, 81% humidity, protein
content around 10% and sugar content around 2%.
[0044] As used herein, "skin" refers to all of the covering tissue
constituting the skin, the mucous membranes and the keratinous
appendages, including hair, nails, eyelashes and eyebrows.
[0045] "Topical application" is understood to be the application or
spreading of a composition containing said white truffle extract,
on the surface of the skin or a mucus membrane.
[0046] What is described herein is a process for obtaining an
extract from grounded white truffle, cosmetic composition
comprising the extract, method of reducing and/or correcting the
signs of aging and photo-aging of the skin by topically applying
the composition comprising white truffle extract to the skin.
[0047] Antioxidants play an important role as health protecting
factors. Scientific evidence suggests that antioxidants reduce the
risk for chronic diseases including cancer and heart disease.
Primary sources of naturally occurring antioxidants are whole
grains, fruits, flower and vegetables. Plant antioxidants are
vitamin C, vitamin E, carotenes, phenolic acid, flavonoids.
[0048] The white truffle extract according to the invention is
known to be rich in polyphenolic compounds, such as phenolic acids.
All these water-soluble molecules known for their antioxidant
activity contribute to provide the antioxidant potent of the white
truffle extract according to the invention. Total polyphenols
content measured is 100 mg/kg of the extract.
[0049] Polyphenolic compounds are recognized to be powerful
antioxidant molecules. "Polyphenolic compounds" are compounds found
abundantly in natural plant food sources that have antioxidant
properties. They refer to all the classes of polyphenols, they mean
compounds comprising at least one diphenol aromatic ring, phenol
group may be optionally etherified or esterified. They can also be
called simply "polyphenol".
[0050] Polyphenols play an important role in maintaining your
health and wellness. Antioxidants as a group help protect the cells
in your body from free radical damage, thereby controlling the rate
at which you age. Antioxidants can be divided into three major
groups: Carotenoids, Allyl sulfides, found in garlic and onions,
Polyphenols (also known as phenolics).
[0051] Polyphenols can be further broken down into four categories:
phenolic acids, flavonoids, lignans, and stilbenes, with additional
subgroupings based on the number of phenol rings they contain, and
on the basis of structural elements that bind these rings to one
another.
[0052] Phenolic acid is a type of phytochemical called polyphenol,
found in a variety of plant based foods; the seeds and skins of
fruits and the leaves of vegetables contain the highest
concentrations. Phenolic acids are readily absorbed through the
walls of the intestinal tract, and they may be beneficial to health
because they work as antioxidants that prevent cellular damage due
to free-radical oxidation reactions. There are many different
phenolic acids found in nature, and they can be divided into two
categories: benzoic acid derivatives, such as gallic acid; and
cinnamic acid derivatives, including caffeic acid and ferulic acid.
The cinnamic acids are more common than the benzoic acids.
[0053] In the present invention "white truffle extract" means the
extract is obtained from the fresh or freezed grounded fruiting
body of the mushroom Tuber magnatum.
[0054] The white truffle extract according to the invention can be
obtained by aqueous extraction. A large number of compounds found
in white truffle extract are likely to have biological activity are
water soluble.
[0055] In a preferred embodiment, the present invention provides a
process for obtaining an extract from white truffle (Tuber
magnatum), said process comprising: [0056] (i) adding water to
grounded white truffle to make a mixture, [0057] (ii) agitating the
said mixture for 2 hours by maintaining temperature from RT to
below 80.degree. C. [0058] (iii) filtering the mixture to remove
the solid part to obtain the extract.
[0059] In a preferred embodiment the grounded white truffle is
macerated in water. The solution is subjected to short time gentle
maceration for 2 hours at a temperature between RT to less than
80.degree. C. Most preferably the temperature is maintained between
RT to 50.degree. C. to preserve the integrity of molecules of
interest such phenolic acids and so their ability to act as
antioxidant.
[0060] The selection of the extraction temperature depends on the
desired type of compounds to be extracted, the structural
characteristics of the botanical source (flowers, fruits, stems,
seeds, leaves, root), the quality and yield required for the
extract, and the economic feasibility for scaling up the process.
The extraction of the phenolic compounds from the plant material is
influenced by the extraction temperature and time, which reflects
the conflicting actions of solubilization and analyte degradation
by oxidation. However, many phenolic compounds are easily
hydrolyzed and oxidized. Long extraction times and high temperature
increase the chance of oxidation of phenolics which decrease the
yield of phenolics in the extracts. The analysis was done at
different temperatures and showed that high temperatures degrade
these types of molecules.
[0061] The water extraction can be carried out at room temperature
or with water heated at no more than 80.degree. C., while being
agitated. Several processes of extraction were tested and selected
as preserving the integrity of molecules present in truffles, with
an acceptable yield while removing the volatile flavoured molecules
responsible for characteristic truffle odor. The targeted molecules
being fragile, it had been shown that the extraction at 50.degree.
C. best met the needs of the invention. Thus preferably, the
extraction is carried out by maceration in water heated at
50.degree. C. for 2 hours. The raw solution is then subjected to
grid filtering to remove insoluble material. After grid filtering,
the aqueous or liquid fraction is collected. To remove smaller
residues of the aqueous extract, a filtration by any process well
known by someone skilled in the art may be carried out.
[0062] In a preferred embodiment, the purification process begins
by successive filtrations using filters with decreasing porosity
from about 50 to about 20 .mu.m until about 0.5-0.2 .mu.m to get an
extract. Preferably, the filters have a decreasing porosity of 50
to 20 .mu.m until t 0.5 .mu.m to 0.2 .mu.m.
[0063] In a preferred embodiment, the purification process is
followed by a double purification by activated carbone.
[0064] In another preferred embodiment, the extract obtained is
composed of protein fragments and peptides with a molecular weight
of less than 60 kDa, as demonstrated by sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE). The obtained extract
is a clear and brilliant solution.
[0065] In another preferred embodiment, the extract is then diluted
at a concentration between 0.5 g/Kg and 1.5 g/Kg, preferably at 1
g/Kg of dry matter with solvents, such as water, glycerol, ethanol,
propanediol, butylene glycol, dipropylene glycol, ethoxylated or
propoxylated glycols, cyclic polyols or any mixture of these
solvents for example 30% butylene glycol and 2% of 1.2 hexanediol
by maintaining the pH between 4 to 5.
[0066] Then, the diluted white truffle extract is sterilized by
sterile filtration. Then the solution is heated overnight at
65.degree. C. to perform low-temperature pasteurization.
[0067] Furthermore, the diluted white truffle extract diluted at a
concentration between 0.5 g/Kg and 1.5 g/Kg, of dry matter with
solvents can be qualitatively and quantitatively analyzed. The
characteristics are the following: [0068] Proteins: 0.05-0.15 g/kg,
[0069] Sugars: 0.35-1.05 g/kg, [0070] Amino acids: 0.025-0.075
g/kg, [0071] Phenolic compounds: 0.05-0.15 g/kg, [0072] The extract
does not contain any ceramide or sphingolipid.
[0073] Protein contents of the white truffle extract have been
determined by Lowry protein assay (Lowry O H, Rosebrough N J, Farr
A L, Randall R J (1951). "Protein measurement with the Folin phenol
reagent", J. Biol. Chem. 193 (1): 265-75) which has been used to
quantify total protein content of the extract. The Lowry assay is a
biochemical assay for determining the total level of protein in a
solution. The Lowry method is based on the reaction of Cu+,
produced by the oxidation of peptide bonds, with Folin-Ciocalteu
reagent. The absorbance of the sample is read on the
spectrophotometer at 550 nm. The protein content is determined
using a BSA standard curve.
[0074] Aminoacid content of the white truffle extract have been
determined starting from a protocol published by Moore et al.
(Moore et al, "Photometric ninhydrin method for use in the
chromatography of amino acids", Journal of Biological Chemistry
1948 Vol. 176 pp. 367-388). The free amino acid content of the
extract was assessed by the formation of a colored complex,
following the rupture of the amine and carboxylic functions by the
reagent ninhydrin. The absorbance of the complex is read on the
spectrophotometer at 570 nm. The total amino acids content is
determined using a standard curve of amino acids pool.
[0075] Total sugars content in the white truffle extract was
determined by colorimetry via an adaptation of the assay described
by (Dubois et al. ("Colorimetric Method for Determination of Sugars
and Related Substances", Anal. Chem., 1956, 28 (3), 350-356). This
analysis consists in the dissolution of the raw material in
concentrated sulfuric acid and then reacting with phenol to form a
colored complex. The absorbance of the complex is read on the
spectrophotometer at 490 nm. The sugar content is determined using
a glucose standard curve.
[0076] Polyphenols content of the white truffle extract was
determined using the Folin-Ciocalteu assay (Singleton et al.
(1999). "Analysis of total phenols and other oxidation substrates
and antioxidants by means of Folin-Ciocalteu reagent", 299: 152).
Polyphenols compounds in the sample react with the Folin-Ciocalteu
reagent, the oxidation of the reagent give a blue color. The
absorbance of the sample is read on the spectrophotometer at 760
nm. The content was expressed as gallic acid equivalents using a
gallic acid standard curve.
[0077] SDS-PAGE electrophoresis was performed to assess molecular
weight of proteins of the extract. The white truffle extract is
heated to 70.degree. C. for 10 minutes in reductive denaturing
conditions in a denaturing sample buffer. An antioxidant solution
is added to the inner chamber (cathode) so that the reduced
proteins do not re-oxidize during electrophoresis. Protein
migration is carried out using the MES running buffer with standard
Novex.RTM. Sharp as a marker for molecular weight. Protein staining
is carried out using silver staining.
[0078] The present application provides a cosmetic composition
comprising white truffle extract obtained by aqueous extraction,
wherein the white truffle extract comprises compounds having a
molecular weight of less than 60 kDa and a physiologically
acceptable medium.
[0079] The advantage of the extract according to the invention is
that small compounds are more stable and reproducible without
having an allergenic effect.
[0080] In another preferred embodiment, the white truffle extract
is present in a concentration range from about 0.01% to about 20%
by weight, preferably 0.1% to about 5% by weight of the total
weight of the composition.
[0081] In another embodiment, the white truffle extract is used for
cosmetic applications, more preferably for topical
applications.
[0082] In another preferred embodiment, the present invention
provides oral, parenteral or topical formulations adapted by the
person skilled in the art, in particular for cosmetic or
dermatological compositions. The compositions according to the
invention are advantageously designed to be administered topically.
These compositions must therefore contain a physiologically
acceptable medium, i.e. compatible with the skin, and cover all
cosmetic or dermatological forms.
[0083] Further, the present compositions preferably are in the form
of an aqueous, hydroalcoholic or oily solution; oil-in-water
emulsion, water-in-oil emulsion or multiple emulsions; creams,
suspensions, powders adapted for application on the skin, mucus
membranes, lips and/or keratinous appendages. These compositions
can also be more or less fluid and have the appearance of a cream,
a lotion, milk, a serum, pomade, a gel, a paste or a mousse. They
can also exist in solid form, as a stick, or can be applied to the
skin as an aerosol. They can also be used as a skincare product
and/or as a makeup product.
[0084] In another embodiment, the composition comprises
conventionally used additive envisaged in the scope of application
as well as necessary additives for their formulation, such as
co-solvents (ethanol, glycerol, benzyl alcohol, damper . . . ),
thickeners, thinners, emulsifiers, antioxidants, colorants, solar
filters, pigments, fillers, preservatives, perfumes, odor
absorbers, essential oils, oligo elements, essential fatty acids,
surfactants, film-forming polymers, chemical filters or minerals,
moisturizing agents or thermal waters etc. Water-soluble,
preferably natural, polymers, such as polysaccharides or
polypeptides, cellulose derivatives of the type methylcellulose or
hydroxypropylcellulose, or even synthetic polymers, poloxamers,
carbomers, siloxanes, PVA or PVP, and in particular polymers sold
by the company Ashland, can be cited, for example.
[0085] It is well understood that the white truffle extract
according to the invention can be used on its own or in conjunction
with other active ingredients.
[0086] Furthermore, the compositions which can be used according to
the invention advantageously contain at least one other active
agent. The following types of ingredients can be cited, in a
non-limiting manner: other peptide active agents, vegetable
extracts, healing agents, anti-aging agents, anti-wrinkle agents,
soothing agents, anti-free radicals, anti-ultraviolet radiation
agents, agents for stimulating dermal macromolecular synthesis or
energetic metabolism, moisturizing agents, antibacterial agents,
antifungal agents, anti-inflammatories, anesthetics, agents
modulating cutaneous differentiation, cutaneous pigmentation or
depigmentation, and agents for stimulating nail and hair
growth.
[0087] In a more specific embodiment, the composition according to
the invention will comprise: [0088] Sunscreens, ultraviolet and
Infra-red screens [0089] Anti-free radical agents, [0090] DHEA
(dehydroepiandrosterone), [0091] At least one cytochrome
co-activating compound, and/or; [0092] One (or more)
aquaporin-activating compound and/or; [0093] One (or more)
sirtuin-activating compound and/or; [0094] One (or more) compound
that increases cell adhesion and/or; [0095] One (or more) compound
that increases the production of matrix proteins of the collagen or
laminin type, etc.; [0096] One (or more) HSP protein-modulating
compound; [0097] One (or more) compound that increases cell energy;
[0098] One (or more) pigmentation-modulating compound such as a
yeast, amaranth, linseed, bean, cacao, corn, soy, sunflower,
rapeseed or pea peptide extract; [0099] One (or more) compound
improving the skin barrier function; [0100] One (or more)
mitochondria-protecting compound. [0101] Vitamin A and notably
retinoic acid, retinol, retinol propionate, retinol palmitate,
[0102] Vitamin B3 and notably niacinamide, nicotinate of
tocopherol, [0103] Vitamin B5, vitamin B6, vitamin B12, panthenol,
[0104] Vitamin C, and notably ascorbic acid, ascorbyl glucoside,
ascorbyl tetrapalmitate, magnesium and sodium ascorbyl phosphate,
[0105] Vitamins E, F, H, K, PP, and coenzyme Q10, Metalloproteinase
inhibitor, activator of Tissue Inhibitor Metalloproteinase (TIMP),
[0106] Aminoacids and notably arginine, ornithine, hydroxyproline,
hydroxyproline dipalmitate, palmitoylglycine, hydroxylysine,
methionine and its derivatives, N-acylated aminoacids, [0107]
Natural or synthetic peptides, including, di-, tri-, tetra-, penta-
and hexapeptides and their lipophilic derivatives, isomers and
complex with other molecules such as metallic ion (i.e. copper,
zinc, manganese, magnesium, and others), peptides sold under
commercial names MATRIXYL.RTM., ARGIRELINE.RTM., CHRONOGEN.TM.,
LAMINIXYL IS.TM., PEPTIDE Q10.TM., COLLAXYL.TM. (patent FR2827170,
ASHLAND.RTM.), PEPTIDE VINCI 01.TM. (patent FR2837098,
ASHLAND.RTM.), PEPTIDE VINCI 02.TM. (patent FR2841781,
ASHLAND.RTM.), ATPeptide.TM. (patent FR2846883, ASHLAND.RTM.) or
synthetic peptide of sequence Arg-Gly-Ser-NH2, sold under
commercial name of ATPeptide.TM. by ASHLAND.RTM.; [0108] Extract of
Artemia salina, sold under commercial name of GP4G.TM. (FR2817748,
ASHLAND.RTM.); [0109] Botanical peptide extracts such as flaxseed
extract (Lipigenine.TM., patent FR2956818, ASHLAND.RTM.), soya
extract, einkorn, grapevine, rapeseed, rice, corn or pea; [0110]
Yeast extracts, such as Dynagen.TM., (patent FR2951946,
ASHLAND.RTM.) or Actopontine.TM. (patent FR2944526, ASHLAND.RTM.);
dehydroacetic acid (DHA), [0111] Natural or synthetic
phystosterols, [0112] alpha- and beta-hydroxyacids, silanols,
[0113] Sugar amines, glucosamine, D-glucosamine,
N-acetyl-glucosamine, N-acetyl-D-glucosamine, mannosamine, N-acetyl
mannosamine, galactosamine, N-acetyl galactosamine, [0114]
Polyphenols, isoflavones, flavonoids, such as grape extract, pine
extract, olive extract, [0115] Lipids such as ceramides or
phospholipids, [0116] Animal oils such as squalenes or squalanes,
[0117] Vegetal oils, such as almond oil, coconut oil, castor oil,
jojoba oil, olive oil, rapeseed oil, peanut oil, sunflower oil,
wheat germ oil, corn germ oil, soybean oil, cotton oil, alfalfa
oil, poppy oil, pumpkin seed oil, evening primrose oil, millet oil,
barley oil, rye oil, safflower oil, passion oil, hazelnut oil, palm
oil, apricot kernel oil, avocado oil, calendula oil, ethoxylated
vegetable oils, or shea butter, the above mentioned compounds can
be natural, such as peptide hydrolysates of plants, or also
synthetic, such as peptide compounds.
[0118] It is clear that the invention is designed for mammals in
general, and more specifically for human beings. The inventors have
indeed identified biological activities which are useful to reduce
and/or correct the cutaneous signs of aging and photo-aging of the
skin and to lighten the skin.
[0119] In yet another embodiment, the present application provides
a cosmetic method for reducing and/or correcting the signs of aging
and photo-aging of the skin, comprising topically applying to the
skin, a composition comprising a white truffle extract according to
the application. Preferably this cosmetic method comprises applying
to the skin, a composition comprising a white truffle extract
wherein the compounds have a molecular weight of less than 60 kDa
in a physiologically acceptable medium.
[0120] In yet another aspect, the present application provides a
cosmetic method to lighten the skin, wherein a cosmetic composition
comprising a white truffle extract according to the invention is
applied topically on the skin which is to be treated. Preferably
this cosmetic method comprises applying to the skin, a composition
comprising a white truffle extract wherein the compounds have a
molecular weight of less than 60 kDa in a physiologically
acceptable medium.
[0121] The embodiments which are specific to this cosmetic method
also result from the above description.
[0122] Further advantages and characteristics of the invention can
be seen in greater detail by reading the illustrative, non-limiting
examples provided.
Example 1: Preparation of a White Truffle Extract (Tuber
magnatum)
[0123] The white truffles (fruiting body) are obtained from Alba in
Italy. The truffle 50 g of grounded freezed white truffle is placed
in 1 liter of distillated water. The solution is heated 2 hours at
temperature of 50.degree. C. Then a filtration 20-50 .mu.m is
carried out to separate solid truffle residue from the liquid part
is carried out. The purification process begins by successive
filtrations using filters with decreasing porosity from 50-20 .mu.m
until 0.3-0.5 .mu.m then 0.25% of activated carbon (Cabot Norit
SXplus) is added to the solution and mixed during 30 minutes at
50.degree. C.; the charcoal is then removed with a 0.3-0.5 .mu.m
filtration; another 0.25% of activated carbon (Cabot Norit SXplus)
is added to the solution and mixed during 30 minutes at 50.degree.
C.; the charcoal is then removed with a 0.3-0.5 .mu.m filtration.
The filtrate is then diluted to obtain an extract having between
0.5-1.5 g/Kg dry matter with 30% butylene glycol and 2%
hexanediol.
[0124] The pH of the solution is adjusted between 4 and 5 to
increase the stability of the extract. After clarification and
dilution, the filtrate is then filter-sterilized with 0.2 .mu.m
filter porosity under sterile condition. White truffle extract was
analyzed using standard procedure. The characteristics of the white
truffle extract obtained are the following: dry matter: 1
g/kg--proteins: 0.1 g/kg--sugars: 0.70 g/kg--amino acids: 0.01 g/kg
and polyphenolic compounds 0.1 g/Kg.
[0125] Proteins content of the white truffle extract have been
determined by Lowry protein assay (Lowry et al, 1951) to quantify
total protein content of the extract. The Lowry assay is a
biochemical assay for determining the total level of protein in a
solution. The Lowry method is based on the reaction of Cu+,
produced by the oxidation of peptide bonds, with Folin-Ciocalteu
reagent. The absorbance of the sample is read on the
spectrophotometer at 550 nm. The protein content was determined
using a BSA (Bovine Serum albumin) standard curve. Aminoacid
content of the extract have been determined starting from a
protocol published by Moore et al (1948), the free amino acid
content of the extract was assessed by the formation of a colored
complex, following the rupture of the amine and carboxylic
functions by the reagent ninhydrin. The absorbance of the complex
is read on the spectrophotometer at 570 nm. The total amino acids
content was determined using a standard curve of amino acids
pool.
[0126] Total sugar content on the extract was determined
colorimetrically via an adaptation of the assay described by Dubois
et al (1956) (Dubois et al, "Colorimetric Method for Determination
of Sugars and Related Substances", Anal. Chem., 1956, 28 (3),
350-356). This analysis consists in the dissolution of the raw
material in concentrated sulfuric acid and then reacting with
phenol to form a colored complex. The absorbance of the complex is
read on the spectrophotometer at 490 nm. The sugar content is
determined using a glucose standard curve.
[0127] Polyphenol content of the white truffle extract was
determined using the Folin-Ciocalteu assay (Singleton et al.,
"Analysis of total phenols and other oxidation substrates and
antioxidants by means of folin-ciocalteu reagent", 1999, 299: 152).
Polyphenol compounds in the sample react with the Folin-Ciocalteu
reagent, the oxidation of the reagent gives a blue color. The
absorbance of the sample is read on the spectrophotometer at 760
nm. The content was expressed as gallic acid equivalents using a
gallic acid standard curve
[0128] SDS PAGE electrophoresis was performed to assess molecular
weight of proteins of the extract. The white truffle extract is
heated to 70.degree. C. for 10 minutes in reductive denaturing
conditions in a denaturing sample buffer. An Antioxidant solution
is added to the inner chamber (cathode) so that the reduced
proteins do not re-oxidize during electrophoresis. Protein
migration is carried out using the MES running buffer with standard
Novex.RTM. Sharp as a marker for molecular weight. Protein staining
is carried out using silver staining.
[0129] The extract obtained is composed of peptides with a
molecular weight of less than 60 kDa.
Example 2: Preparation of a White Truffle Extract (Tuber
magnatum)
[0130] Briefly, the white truffles (fruiting body) are obtained
from Alba in Italy. 100 g of freezed grounded white truffle is
placed in 1 liter of distillated water. Solution pH is adjusted to
2 with chlorhydric acid. The solution is heated 2 hours at
temperature of 80.degree. C. Then a filtration 20-50 is carried out
to separate solid truffle residue from the liquid part. The
purification process begins by successive filtrations using filters
with decreasing porosity from 50-20 .mu.m until 0.3-0.5 .mu.m then
1% of activated carbon (Cabot Norit SXplus) is added to the
solution and mixed during 30 minutes at 50.degree. C.; the charcoal
is then removed with a 0.3-0.5 .mu.m filtration. The filtrate
obtained is 20 g/Kg dry matter and is then diluted to obtain an
extract having between 0.5-1.5 g/Kg dry matter. The pH of the
solution is adjusted between 4 and 5 to increase the stability of
the extract. After clarification and dilution, the filtrate is then
filter-sterilized with 0.2 .mu.m filter porosity under sterile
condition.
Example 3: Preparation of a White Truffle Extract (Tuber
magnatum)
[0131] Briefly, the white truffles (fruiting body) are obtained
from Alba in Italy. 100 g of freezed grounded white truffle is
placed in 1 liter of distillated water. Solution pH is adjusted to
2 with chlorhydric acid. The solution is heated 2 hours at
temperature of 80.degree. C. Then a filtration 20-50 .mu.m is
carried out to separate solid truffle residue from the liquid part.
The purification process begins by successive filtrations using
filters with decreasing porosity from 50-20 .mu.m until 0.3-0.5
.mu.m then 0.25% of activated carbon (Cabot Norit SXplus) is added
to the solution and mixed during 30 minutes at 50.degree. C.; the
charcoal is then removed with a 0.3-0.5 .mu.m filtration; another
0.25% of activated carbon (Cabot Norit SXplus) is added to the
solution and mixed during 30 minutes at 50.degree. C.; the charcoal
is then removed with a 0.3-0.5 .mu.m filtration. The filtrate
obtained is 20 g/kg dry matter and is then diluted to obtain an
extract having between 0.5-1.5 g/Kg dry matter. The pH of the
solution is adjusted between 4 and 5 to increase the stability of
the extract. After clarification and dilution, the filtrate is then
filter-sterilized with 0.2 .mu.m filter porosity under sterile
condition.
Example 4: Preparation of a White Truffle Extract (Tuber
magnatum)
[0132] Briefly, the white truffles (fruiting body) are obtained
from Alba in Italy. 100 g of freezed grounded white truffle is
placed in 1 liter of a solution composed of 10 mM tetrasodic EDTA.
The solution is heated 2 hours at temperature of 50.degree. C. Then
a filtration 20-50 .mu.m is carried out to separate solid truffle
residue from the liquid part. The purification process begins by
successive filtrations using filters with decreasing porosity from
50-20 .mu.m until 0.3-0.5 .mu.m, then 0.25% of activated carbon
(Cabot Norit SXplus) is added to the solution and mixed during 30
minutes at 50.degree. C.; the charcoal is then removed with a
0.3-0.5 .mu.m filtration; another 0.25% of activated carbon (Cabot
Norit SXplus) is added to the solution and mixed during 30 minutes
at 50.degree. C.; the charcoal is then removed with a 0.3-0.5 .mu.m
filtration. The filtrate obtained is 20 g/kg dry matter and is then
diluted to obtain an extract having between 0.5-1.5 g/Kg dry
matter. The pH of the solution is adjusted between 4 and 5 to
increase the stability of the extract. After clarification and
dilution, the filtrate is then filter-sterilized with 0.2 .mu.m
filter porosity under sterile condition.
Example 5: Evaluation of the White Truffle on Skin Aging by
Extracellular Matrix Evaluation on Fibroblasts
[0133] The purpose of this study is to show the effect of the White
Truffle extract on aging by the extracellular matrix (ECM)
evaluation, regarding collagen I expression.
[0134] Protocol:
[0135] Normal human fibroblasts were treated twice a day for 48
hours with a solution of White Truffle extract, according to
example 1, 2, 3 or 4, diluted at 1/200 eme in the culture medium,
leading to a final concentration of 0.5% vol/vol.
[0136] For immunolabelling by anti-collagen I antibody, the cells
were washed and fixed with cold methanol. The cells were then
incubated in the presence of a specific anti-collagen I antibody
(Tebu, ref. 600-401-103-0.5, rabbit polyclonal), and then a
secondary suitable antibody, coupled with a fluorescent dye. After
mounting in a particular medium, the slides were observed by
epifluorescence microscope (Zeiss Axiovert 200M microscope).
Fluorescence intensity was quantified by analyzing the image using
Volocity.RTM. 6.3. software (PerkinElmer, Inc.).
[0137] Results:
[0138] Only the treatments with the White Truffle extract according
to example 1 diluted at 0.5% for 48 hours showed a highly
significant increase (Student's t-test) in collagen I expression on
fibroblasts. The other extract application didn't show any
efficacy.
[0139] The results are illustrated in FIG. 1.
[0140] Conclusions:
[0141] White Truffle extract at 0.5%, through the stimulation of
collagen I, improved the extracellular matrix on fibroblasts. The
extract according to example 1 gave the best results.
Example 6: Evaluation of the White Truffle Extracts on Lightening,
by Melanin Content Evaluation on Ex Vivo Skin Biopsies
[0142] The purpose of this study is to show the effect of the White
Truffle extract on lightening by melanin content evaluation using
Fontana-Masson staining. The Fontana-Masson staining is based on
the melanin ability to reduce solutions of ammoniacal silver
nitrate to metallic silver (brown) without the use of an external
reducing agent.
[0143] Protocol:
[0144] Normal human skin biopsies of 6 mm of diameter were
maintained ex vivo in a specific culture medium (DMEM at 1 g/L,
HAMF12, fetal calf serum and antibiotics). Biopsies were treated
twice a day for 48 hours with a solution of White Truffle extract,
according to example 1, 2, 3 or 4 diluted at 1/200 eme in PBS,
leading to a final concentration of 0.5% vol/vol, respectively. The
control condition is performed by applying PBS 1.times..
[0145] For Fontana-Masson staining, tissues were fixed and embedded
in paraffin. Embedded skin biopsies were then cut and sections were
deparaffinized and rehydrated. Then, a stock solution containing
ammonium hydroxide and silver nitrate was added on each section.
After a distilled water wash, biopsies were incubated with 5%
sodium thiosulfate and washed again. After mounting in a particular
medium, the slides were examined using an Eclipse E600 microscope
(Nikon). The number of brown/dark pixels was quantified by
analyzing the image using ImageJ software.
[0146] Results:
[0147] The treatments with the White Truffle extract according to
examples 1 and 4 diluted at 0.5% for 48 hours showed a significant
decrease (Student's t-test) of melanin content on ex vivo skin
biopsies. The other extract application didn't show any
efficacy.
[0148] The results are illustrated in FIG. 2. It was observed that
the extract according to ex. 1 gave better results than the extract
according to ex. 4.
[0149] Conclusions:
[0150] White Truffle extract application at 0.5% showed a
lightening efficacy by decreasing melanin content on ex vivo skin
biopsies. The extract according to example 1 gave the best
results.
[0151] The extract prepared according to example 1 was the only to
act on both extracellular matrix and lightening pathways. It was
selected as the best candidate.
Example 7: Evaluation of the White Truffle Extract According to
Example Ion Autophagy Pathway, on Keratinocytes and Ex Vivo Skin
Biopsies
[0152] The purpose of this study is to show the effect of the White
Truffle extract on autophagy. Autophagy is a catabolic process for
the autophagosomic-lysosomal degradation of bulk cytoplasmic
contents (Cuervo A M et al. "Autophagy, nutrition and immunology",
Mol Aspects Med. 33(1):2-13, 2012). Here, LC3
(Microtubule-associated protein 1A/1B-light chain 3) which is a
structural protein implicated in the formation of the
autophagosomes (Melendez A and Levine B. "Autophagy in C. elegans",
WormBook. 24:1-26, 2009) was evaluated.
[0153] Protocol:
[0154] Evaluation on Keratinocytes
[0155] Normal human keratinocytes were treated twice a day for 48
hours with a solution of White Truffle extract, according to
example 1, diluted at 1/200 eme in the culture medium, leading to a
final concentration of 0.5% vol/vol.
[0156] For immunolabelling by LC3 antibody, the cells were washed
and fixed with cold methanol. The cells were then incubated in the
presence of a specific LC3 antibody (Cell Signaling, ref. PM036,
rabbit polyclonal), and then a secondary suitable antibody, coupled
with a fluorescent dye. After mounting in a particular medium, the
slides were observed by epifluorescence microscope (Zeiss Axiovert
200M microscope). Fluorescence intensity was quantified by
analyzing the image using Volocity.RTM. 6.3. software (PerkinElmer,
Inc.).
[0157] Evaluation on Ex Vivo Skin Biopsies
[0158] Normal human skin biopsies of 6 mm of diameter were
maintained ex vivo in a specific culture medium (DMEM at 1 g/L,
HAMF12, fetal calf serum and antibiotics). Biopsies were treated
twice a day for 48 hours with a solution of White Truffle extract,
according to example 1, diluted at 1/200 eme in PBS, leading to a
final concentration of 0.5% vol/vol, respectively. The control
condition is performed by applying PBS IX.
[0159] For immunolabelling by LC3 antibody, tissues were fixed and
embedded in paraffin. Embedded skin biopsies were then cut and
sections were deparaffinized and rehydrated. Then, an unmasking
protocol was performed before applying a specific anti-LC3 antibody
(Cell Signaling, PM036, rabbit polyclonal), and then a secondary
suitable antibody, coupled with a fluorescent dye. After mounting
in a particular medium, the slides were observed by epifluorescence
microscope (Zeiss Axiovert 200M microscope).
[0160] Results:
[0161] The treatments with a solution of White Truffle extract
diluted at 0.5% for 48 hours showed a highly significant increase
(Student's t-test) in LC3 expression both on keratinocytes and ex
vivo skin biopsies.
[0162] Conclusion:
[0163] White Truffle extract at 0.5%, through the stimulation of
LC3, improved the autophagy pathway.
* * * * *